Determination of KRN321 in Rat Serum by ELISA and Correlation Between ELISA and in vitro Bioassay

Abstract

KRN321, darbepoetin alfa, is a hyperglycosylated analog of recombinant human erythropoietin (rHuEPO, epoetin alfa). We carried out the validation study by using a commercially available ELISA assay kit to establish the ELISA quantitation method for KRN321 in rat serum for the implementation of the pharmacokinetic studies with a lower limit of quantitation at 100 pg/mL and quantitation range from 100 to 4,000 pg/mL. We also established the in vitro bioassay method as an index of biological activity using UT‐7/Epo, derived from a human leukemia cell line with a lower limit of quantitation, at 10 ng/mL. Furthermore, good correlation was observed between the two methods; it indicated that KRN321 concentration determined by the ELISA maintained biological activity.

Keywords:

• KRN321
• Darbepoetin alfa
• ELISA
• Bioassay
• Serum
• Validation

Acknowledgments

The authors wish to thank Dr. Takenori Natori from the Pharmaceutical Development Laboratories, Pharmaceutical Division, Kirin brewery Co., Ltd. for continuous and valuable support.